We improved the bispecific antibody platform that primarily engages natural killer (NK) cells to kill cancer cells through antibody-dependent cellular cytotoxicity (ADCC) by adding IL-15 as a ...crosslinker that expands and self-sustains the effector NK cell population. The overall goal was to target B7-H3, an established marker predominantly expressed on cancer cells and minimally expressed on normal cells, and prove that it could target cancer cells in vitro and inhibit tumor growth in vivo. The tri-specific killer engager (TriKE
) was assembled by DNA shuffling and ligation using DNA encoding a camelid anti-CD16 antibody fragment, a wild-type IL-15 moiety, and an anti-B7-H3 scFv (clone 376.96). The expressed and purified cam1615B7H3 protein was tested for in vitro NK cell activity against a variety of tumors and in vivo against a tagged human MA-148 ovarian cancer cell line grafted in NSG mice. cam1615B7H3 showed specific NK cell expansion, high killing activity across a range of B7-H3+ carcinomas, and the ability to mediate growth inhibition of aggressive ovarian cancer in vivo. cam1615B7H3 TriKE improves NK cell function, expansion, targeted cytotoxicity against various types of B7-H3-positive human cancer cell lines, and delivers an anti-cancer effect in vivo in a solid tumor setting.
Clinical studies validated antibodies directed against HER2, trastuzumab, and pertuzumab, as useful methodology to target breast cancer cases where HER2 is expressed. The hope was that HER2 targeting ...using these antibodies in ovarian cancer patients would prove useful as well, but clinical studies have shown lackluster results in this setting, indicating a need for a more comprehensive approach. Immunotherapy approaches stimulating the innate immune system show great promise, although enhancing natural killer (NK) function is not an established mainstream immunotherapy. This study focused on a new nanobody platform technology in which the bispecific antibody was altered to incorporate a cytokine. Herein we describe bioengineered CAM1615HER2 consisting of a camelid VHH antibody fragment recognizing CD16 and a single chain variable fragment (scFv) recognizing HER2 cross-linked by the human interleukin-15 (IL-15) cytokine. This tri-specific killer engager (TriKE
) showed in vitro prowess in its ability to kill ovarian cancer human cell lines. In addition, we demonstrated its efficacy in inducing potent anti-cancer effects in an in vivo xenograft model of human ovarian cancer engrafting both cancer cells and human NK cells. While previous approaches with trastuzumab and pertuzumab faltered in ovarian cancer, the hope is incorporating targeting and cytokine priming within the same molecule will enhance efficacy in this setting.
New treatments are required to enhance current therapies for lung cancer. Mesothelin is a surface protein overexpressed in non-small cell lung cancer (NSCLC) that shows promise as an ...immunotherapeutic target in phase I clinical trials. However, the immunosuppressive environment in NSCLC may limit efficacy of these therapies. We applied time-of-flight mass cytometry to examine the state of circulating mononuclear cells in fourteen patients undergoing treatment for unresectable lung cancer. Six patients had earlier stage NSCLC (I-IVA) and eight had highly advanced NSCLC (IVB). The advanced NSCLC patients relapsed with greater frequency than the earlier stage patients. Before treatment, patients with very advanced NSCLC had a greater proportion of CD14
myeloid cells than patients with earlier NSCLC. These patients also had fewer circulating natural killer (NK) cells bearing an Fc receptor, CD16, which is crucial to antibody-dependent cellular cytotoxicity. We designed a high affinity tri-specific killer engager (TriKE
) to enhance NK cytotoxicity against mesothelin
targets in this environment. The TriKE consisted of CD16 and mesothelin binding elements linked together by IL-15. TriKE enhanced proliferation of lung cancer patient NK cells
. Lung cancer lines are refractory to NK cell killing, but the TriKE enhanced cytotoxicity and cytokine production by patient NK cells when challenged with tumor. Importantly, TriKE triggered NK cell responses from patients at all stages of disease and treatment, suggesting TriKE can enhance current therapies. These pre-clinical studies suggest mesothelin-targeted TriKE has the potential to overcome the immunosuppressive environment of NSCLC to treat disease.
BackgroundThe tumor microenvironment contains stromal cells, including endothelial cells and fibroblasts, that aid tumor growth and impair immune cell function. Many solid tumors remain difficult to ...cure because of tumor-promoting stromal cells, but current therapies targeting tumor stromal cells are constrained by modest efficacy and toxicities. TEM8 is a surface antigen selectively upregulated on tumor and tumor stromal cells, endothelial cells and fibroblasts that may be targeted with specific natural killer (NK) cell engagement.MethodsA Tri-specific Killer Engager (TriKE) against TEM8—‘cam1615TEM8’—was generated using a mammalian expression system. Its function on NK cells was assessed by evaluation of degranulation, inflammatory cytokine production, and killing against tumor and stroma cell lines in standard co-culture and spheroid assays. cam1615TEM8-mediated proliferation and STAT5 phosphorylation in NK cells was tested and compared with T cells by flow cytometry. NK cell proliferation, tumor infiltration, and tumor and tumor-endothelium killing by cam1615TEM8 and interleukin-15 (IL-15) were assessed in NOD scid gamma (NSG) mice.Resultscam1615TEM8 selectively stimulates NK cell degranulation and inflammatory cytokine production against TEM8-expressing tumor and stromal cell lines. The increased activation translated to superior NK cell killing of TEM8-expressing tumor spheroids. cam1615TEM8 selectively stimulated NK cell but not T cell proliferation in vitro and enhanced NK cell proliferation, survival, and tumor infiltration in vivo. Finally, cam1615TEM8 stimulated NK cell killing of tumor and tumor endothelial cells in vivo.ConclusionsOur findings indicate that the cam1615TEM8 TriKE is a novel anti-tumor, anti-stroma, and anti-angiogenic cancer therapy for patients with solid tumors. This multifunctional molecule works by selectively targeting and activating NK cells by costimulation with IL-15, and then targeting that activity to TEM8+ tumor cells and TEM8+ tumor stroma.
Chemotherapy-refractive multiple myeloma (MM) is serious and life-threatening, and better treatments are urgently needed. BCMA is a prominent marker on the cell surface of MM cells, rendering it an ...accepted target for antibody therapy. Considering that MM is a liquid tumor and immunotherapy has enjoyed success against leukemia, we devise an approach designed to enhance NK cell activity against MM. Ordinarily, NK cells function to naturally survey the body and eliminate malignant cells. Our platform approach is designed to enhance NK function. A tri-specific immune-engaging TriKE is manufactured, consisting of a camelid nanobody VHH antibody fragment recognizing CD16 expressed on NK cells and an scFv antibody fragment specifically recognizing BCMA. These two fragments are crosslinked by the human cytokine interleukin-15 (IL-15) known to have prominent activating effects on NK cells. The molecule, when tested by flow cytometry, shows activation of NK cells in their numbers and activity. Additionally, the molecule demonstrates anti-cancer effects in an in vivo xenograft model of human MM. We believe that the drug will have the capability of enhancing NK cells at the site of the immune synapse, i.e., the effector:target cell interface, and this will promote cancer remissions.
BackgroundAcute myeloid leukemia (AML) incidence increases with age. Five year survival for those over 65 is less than 11%, highlighting the need for safe interventions to improve outcomes. Adoptive ...natural killer (NK) cell products have achieved success as a ‘bridge to transplant’ in refractory leukemia and lymphoma, inducing remission to a point where patients are eligible for stem cell transplantation. Multiplexed-engineered induced pluripotent stem cells (iPSCs) are a reproducible source of highly functional NK cells (iNK) for on-demand treatment and broad patient access. Clinical trials are currently testing iNK cells with therapeutic antibodies for the treatment of leukemia and lymphoma (NCT04023071, NCT04614636, NCT04714372). We have developed a protocol for the production of highly functional iNK cells, engineered for greater anti-tumor effect. However, we hypothesized that performing NK cell lineage commitment under physiological oxygen conditions found in bone marrow (5%) would create a niche that could support the generation of a more functional cell product.Methods iPSCs are matured into CD34+ precursors, then differentiated into iNK cells that are subsequently expanded to clinically-relevant quantities (figure 1A). We have previously published on iNK cells that consist of three unique edits: high-affinity non-cleavable CD16, membrane bound IL-15 and knockout of CD38. Using these cells, we performed stage specific differentiation from CD34+ precursors in 5% oxygen (‘physoxic iNK’) or conventional 20% oxygen, with subsequent expansion in 20% oxygen. iNK cells were compared for their phenotype (CyTOF), proliferation (flow cytometry), cytotoxicity (live cell imaging), metabolic stability (reactive oxygen species staining by flow cytometry) and ability to control tumor (xenograft mouse model).ResultsCyTOF analysis revealed a more naive phenotype in physiological oxygen conditions that persisted after expansion. However, these cells were equally capable of natural cytotoxicity and antibody-dependent cellular cytotoxicity. In a xenograft model of AML (NSG mice with HL60-GFP/luciferase; figure 1B) there was greater persistence of physoxic iNK cells in blood and bone marrow (figure 1C), correlating with greater tumor control within the bone marrow (figure 1D) and across the whole animal (figure 1E). When exposed to oxidative stress, physoxic iNK cells were more resilient, with lower reactive oxygen species detected in their mitochondria, suggesting greater tumor control arose from greater persistence within the animal, rather than better cytotoxicity.ConclusionsThese data suggest that manufacturing therapeutic NK cells in a physiological environment at a unique stage of lineage commitment can generate resilient cells with a greater durability for anti-tumor activity.AcknowledgementsThis project was supported by grants from the Department of Defense (CA200922) and National Institutes of Health (R35 CA197292, P01 CA111412). Parts of the figure were drawn using images from Servier Medical Art and from Biorender.Ethics ApprovalThe University of Minnesota human research protection program determined this was not human research (institutional review board ID: STUDY00013106)Abstract 365 Figure 1Differentiating NK cells in 5% oxygen, compared to 20% oxygen, improves their persistence and control of tumor in a xenograft model of AML. (A) The manufacturing process for iNK cells. iPSC-derived iNK cells were differentiated in 5% or 20% oxygen at the indicated stage of lineage commitment. (B) Schematic representation of xenograft mouse study for AML. A single dose of iNK cells was delivered intravenously on day l. Blood was taken to analyze circulating cells by flow cytometry; bioluminescent imaging tracked tumor load for HL60-luciferase/GFP cells; bone marrow and spleens were harvested on day 21 and analyzed by flow cytometry (C) Physoxic iNK cells are found in mouse bone marrow in greater numbers than conventional iNK cells three weeks after intravenous injection. Bars show the median and interquartile range. Mann Whitney test. (D) Less HL60 xenograft tumor is found in mouse bone marrow when mice are treated with physoxic iNK cells compared to conventional iNK cells (day 21). Bars show the median and interquartile range. Mann Whitney test. (E) Less HL60 xenograft tumor is detected by bioluminescent imaging when mice are treated with physoxic iNK cells compared to conventional iNK cells. Bars show the mean. Each dot represents an animal. Two way ANOVA with Dunnett’s multiple comparison test. One of two independent experiments is shown. * p<0.05. ** p<0.01, *** p<0.001.
BackgroundB7H3 is a tumor associated antigen (TAA), found on numerous malignancies including prostate, lung, and breast cancers. High levels of B7H3 expression are correlated with late stage disease ...and poor prognosis. Furthermore, B7H3 is minimally expressed on normal tissue, making it an ideal TAA for broad cancer treatment strategy. We developed a tri-specific killer engager (TriKETM) consisting of a nanobody anti-CD16, IL-15, and nanobody anti-B7H3 joined by flexible linkers (camB7H3 TriKE) (figure 1A). The combination of B7H3 TriKE with an off-the-shelf NK cell therapy presents an appealing therapeutic strategy for the treatment of solid tumors with decreased risk of toxicity in allogeneic settings compared to T-cell derived products.MethodsAn anti-B7H3 nanobody was developed via biopanning and cloned into a TriKE vector. TriKE was produced in Expi293 cells and affinity purified using poly-His tag. NK cells were co-incubated with cell lines exhibiting a range of B7H3 expression and with 3nM of camelid B7H3 TriKE or control. We have previously derived NK cells expressing high affinity non-cleavable hnCD16, CD38 KO, and IL-15/IL-15R fusion from clonal master engineered iPSC lines. Engineered iNK cells were tested in conjunction with the TriKE. A repeated measures ANOVA was used for statistical comparisons as noted in figure legendsResultsEngineered iNK cells co-incubated with camB7H3 TriKE and C4-2 prostate cells significantly increased degranulation (CD107a) and cytokine production (IFN-gamma) compared to controls (figure 1B/C, P<0.05, n=3). camB7H3 TriKE directly bound C4-2 cells with an estimated EC50 of approximately 3nM. camB7H3 TriKE increased percentages of engineered iNK cells dividing robustly (3 or more times) compared to corresponding IL-15 doses at 3 nM (figure 1D, P<0.001, n=3). Furthermore, camB7H3 TriKE enhanced cytotoxic activity of engineered iNK cells against a variety of tumor cells in 2D and spheroid format independent of cytokine support (figure 1E-F). Engineered iNK cells incorporating an anti-B7H3 chimeric antigen receptor (CAR) is also being developed and will be discussed.Abstract 470 Figure 1A) Schematic of TriKE molecule demonstrating spatial relationship of anti-CD16 nanobody, IL-15, and anti-B7H3 nanobody with flexible linker regions. B) Percent of PB NK cells CD107a as a marker of NK degranulation. Unselected PBMCs were stimulated with 3nM camB7H3 TriKE, 3nM IL-15, or no treatment with B7H3-expressing C4-2 prostate cancer cell lines Cells were stimulated for 5 hours and evaluated for degranulation (surface CD107a) by flow cytometry, gating on the NK (CD56+CD3-) population (displayed). Graphs display mean ± SEM. P<0.05 using repeated measures ANOVA. C) Percent of PB NK cells expressing intracellular interferon-gamma. Unselected PBMCs were stimulated with 3nM camB7H3 TriKE, 3nM IL-15, or no treatment with B7H3-expressing C4-2 prostate cancer cell lines Cell were stimulated for 5 hours and evaluated for degranulation (surface CD107a) by flow cytometry, gating on the NK (CD56+CD3-) population (displayed). Graphs display mean ± SEM. P<0.05 using repeated measures ANOVA. D) Percent of PB NK cells dividing robustly (3 or more times) over a 7 day stimulation with 3nM camB7H3 TriKE, 3nM IL-15, or no treatment. PBMCs were incubated with Cell-Trace Violet reagent prior to stimulation. Graphs display mean ± SEM. P<0.001 using repeated measures ANOVA. E) Representative 2D IncuCyte images of PC3 prostate cancer cell lines transduced with NucLight Red. Cells were co-incubated with iNK alone, iNK with 3nM IL-15 or iNK with 3 nM antiB7H3 TriKE. Images represent remaining NucLight Red transduced PC3 cells after 72 hours of co-incubation as noted above. F) Representative microspheriod IncuCyte images of PC3 prostate cancer lines transduced with NucLight Red. Cells were co-incubated with iNK cells alone, iNK with 3 nM IL-15 or iNK with 3 nM antiB7H3 TriKE. Images represent remaining NucLight Red transduced PC3 cells after 72 hours of co-incubation as noted aboveConclusionscamB7H3 TriKE dramatically increases function and activation on endogenous NK cells as well as engineered iNK cell, which can be adoptively transferred to patients with a broad range of cancers, including prostate cancer. TriKE activity was potent across a broad concentration spectrum and corresponded directly with B7H3 target expression. These studies represent the proof-of-concept of a novel pairing of off-the-shelf, engineered iNK cells with B7H3-directed pan-cancer engager molecules (TriKEs and CARs) to enhance specificity, persistence and anti-tumor function.
Select subsets of immune effector cells have the greatest propensity to mediate antitumor responses. However, procuring these subsets is challenging, and cell-based immunotherapy is hampered by ...limited effector-cell persistence and lack of on-demand availability. To address these limitations, we generated a triple-gene-edited induced pluripotent stem cell (iPSC). The clonal iPSC line was engineered to express a high affinity, non-cleavable version of the Fc receptor CD16a and a membrane-bound interleukin (IL)-15/IL-15R fusion protein. The third edit was a knockout of the ecto-enzyme CD38, which hydrolyzes NAD+. Natural killer (NK) cells derived from these uniformly engineered iPSCs, termed iADAPT, displayed metabolic features and gene expression profiles mirroring those of cytomegalovirus-induced adaptive NK cells. iADAPT NK cells persisted in vivo in the absence of exogenous cytokine and elicited superior antitumor activity. Our findings suggest that unique subsets of the immune system can be modeled through iPSC technology for effective treatment of patients with advanced cancer.
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•iADAPT NK cells and adaptive NK cells share metabolic and transcriptional features•iADAPT NK cells are cytokine autonomous•iADAPT NK cells display enhanced innate immune function
Optimized iPSC-derived NK (iNK) cells have been developed for on-demand cancer immunotherapy. Here, Cichocki and colleagues describe a triple-gene-edited iNK cell product, termed iADAPT NK, which persists and functions in vivo in the absence of exogenous cytokines and can be combined with therapeutic antibodies for enhanced tumor targeting.
Natural killer (NK) cells are lymphocytes well suited for adoptive immunotherapy. Attempts with adoptive NK cell immunotherapy against ovarian cancer have proven unsuccessful, with the main ...limitations including failure to expand and diminished effector function. We investigated if incubation of NK cells with interleukin (IL)-12, IL-15, and IL-18 for 16h could produce cytokine-induced memory-like (CIML) NK cells capable of enhanced function against ovarian cancer.
NK cells were preactivated briefly with IL-12, IL-15, and IL-18, rested, then placed against ovarian cancer targets to assess phenotype and function via flow cytometry. Real-time NK-cell-mediated tumor-killing was evaluated. Using ascites cells and cell-free ascites fluid, NK cell proliferation and function within the immunosuppressive microenvironment was evaluated in vitro. Finally, CIML NK cells were injected intraperitoneal (IP) into an in vivo xenogeneic mouse model of ovarian cancer.
CIML NK cells demonstrate enhanced cytokine (IFN-γ) production and NK-cell-mediated killing of ovarian cancer. NK cells treated overnight with cytokines led to robust activation characterized by temporal shedding of CD16, induction of CD25, and enhanced proliferation. CIML NK cells proliferate more with enhanced effector function compared to controls in an immunosuppressive microenvironment. Finally, human CIML NK cells exhibited potent antitumor effects within a xenogeneic mouse model of ovarian cancer.
CIML NK cells have enhanced functionality and persistence against ovarian cancer in vitro and in vivo, even when exposed to ascites fluid. These findings provide a strategy for NK cell-based immunotherapy to circumvent the immunosuppressive nature of ovarian cancer.
•Cytokine-induced memory-like (CIML) NK cells demonstrate enhanced function and proliferation against ovarian cancer cells.•A novel killing assay demonstrated enhanced CIML NK-cell-mediated cytolytic function against ovarian cancer in real time.•The functional and proliferative ability within immunosuppressive ascites was rescued with healthy donor CIML NK cells.•Human CIML NK cells exhibit potent antitumor effects within an in vivo mouse model of ovarian cancer.
•iDuo NK cells prevent antigen escape by targeting both CD19+ and CD19− lymphoma cells.•iDuo NK cells demonstrate enhanced anti-tumor activity and persistence without the need for cytokine support.
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Substantial numbers of B cell leukemia and lymphoma patients relapse due to antigen loss or heterogeneity after anti-CD19 chimeric antigen receptor (CAR) T cell therapy. To overcome antigen escape and address antigen heterogeneity, we engineered induced pluripotent stem cell-derived NK cells to express both an NK cell-optimized anti-CD19 CAR for direct targeting and a high affinity, non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity. In addition, we introduced a membrane-bound IL-15/IL-15R fusion protein to promote in vivo persistence. These engineered cells, termed iDuo NK cells, displayed robust CAR-mediated cytotoxic activity that could be further enhanced with therapeutic antibodies targeting B cell malignancies. In multiple in vitro and xenogeneic adoptive transfer models, iDuo NK cells exhibited robust anti-lymphoma activity. Furthermore, iDuo NK cells effectively eliminated both CD19+ and CD19− lymphoma cells and displayed a unique propensity for targeting malignant cells over healthy cells that expressed CD19, features not achievable with anti-CAR19 T cells. iDuo NK cells combined with therapeutic antibodies represent a promising approach to prevent relapse due to antigen loss and tumor heterogeneity in patients with B cell malignancies.
Cichocki et al report on a novel CAR-natural killer (CAR-NK) cell therapy for B-cell lymphoma that addresses antigen loss, tumor heterogeneity, and failed CAR-cell persistence. The authors engineered induced pluripotent stem cell (iPSC)-derived NK cells with anti-CD19 CAR and a noncleavable CD16 that augments toxicity in the presence of anti-CD20 therapy and added an IL-15/IL-15R fusion to promote cytokine-independent persistence. In vitro and xenograft models treated with the engineered NK cells plus rituximab had robust activity and may represent a promising “off the shelf” approach to targeting B-cell malignancies.
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